It is well established that genetic and epigenetic alterations play important roles in the initiation and progression of colorectal cancer (CRC). A major challenge to our understanding of how this process occurs is to understand how these genetic and epigenetic changes work pathologically to allow for uncontrolled cancer cell growth. Until now, the systematic dissection of the signaling pathways and genes that control CRC initiation and progression have been limited by the use of primary human tumor samples, lack of suitable long-term culture systems, and mouse models that do not accurately recapitulate this process. The recent development of a robust long-term primary colonic epithelium culture system by our group that is capable of modeling tumorigenesis affords the opportunity to begin to explore the role of differential sequence of oncogene and tumor suppressor gene activation or inactivation, respectively, and the functional evaluation of CRC-upregulated or downregulated microRNAs in the development of CRC. Specifically, this proposal seeks to address the hypothesis that the sequence in which one acquires genetic alterations alters CRC progression, and possibly clinical outcomes. Likewise, this proposal seeks to functionally validate the role of microRNAs that are differentially expressed in CRC vs. normal human colonic epithelium. The studies proposed herein will facilitate our fundamental understanding of colon cancer initiation and progression, allowing for the development of more efficacious and targeted approaches to therapy. PUBLIC HEALTH RELEVANCE: Colorectal Cancer is a diverse and heterogeneous disease that is caused by the progressive accumulation of genetic and epigenetic alterations. Health disparities and diverse clinical outcomes can be partially attributed to the specific genetic alteration that occurs within a specific tumor. Understanding how alterations in gene function, or the sequence in which one acquires them, and how changes in microRNA expression contribute to colorectal cancer development will provide novel insights that will contribute to our ability to develop better treatments, therapies, and/or diagnostic tools.